The present invention relates to isolation of cytotoxic T lymphocyte (CTL) clones. In particular, the present invention relates to isolated CTL clones that are specific for MAGE-1 and MAGE-4, respectively. The CTL clones of the present invention have been isolated by successive steps of stimulation and testing of lymphocytes with antigen presenting cells which present antigens derived from different expression systems, e.g., from recombinant Yersinia, recombinant Salmonella, or recombinant viruses. The present invention further relates to the MAGE-1 and MAGE-4 antigenic peptides as well as the peptide/HLA complexes which are recognized by the isolated CTL clones.
An important facet of the immune response in a mammalian subject is the recognition by T cells of the complexes of the cell surface molecules, i.e., the complexes of peptides and HLA (human leukocyte antigens) or MHC (major histocompatibility complexes) molecules. These peptides are derived from larger molecules which are processed by the cells which also present the HLA/MHC molecules. See in this regard, Male et al., Advanced Immunology (J. P. Lipincott Company, 1987), especially chapters 6-10. The interaction between T cell and HLA/peptide complexes is restricted, requiring a T cell specific for a particular combination of an HLA molecule and a peptide. If a specific T cell is not present, there is no T cell response even if its partner complex is present. Similarly, there is no response if the specific complex is absent, but the T cell is present. This mechanism is involved in the immune system response to foreign materials, in autoimmune pathologies, and in responses to cellular abnormalities.
Most progressively growing neoplastic cells express potentially immunogenic tumor-associated antigens (TAAs), also called tumor rejection antigens (TRAs). A number of genes have been identified that encode tumor rejection antigen precursors (or TRAPs), which are processed into TRAs in tumor cells. Such TRAP-encoding genes include members of the MAGE family, the BAGE family, the DAGE/Prame family, the GAGE family, the RAGE family, the SMAGE family, NAG, Tyrosinase, Melan-A/MART-1, gp 100, MUC-1, TAG-72, CA125, mutated proto-oncogenes such as praise, mutated tumor suppressor genes such as p53, tumor associated viral antigens such as HPV16 E7. See, e.g., review by Van den Eynde and van der Bruggen (1997) in Curr. Opin. Immunol. 9:684-693, Sahin et al. (1997) in Curr. Opin. Immunol. 9:709-716, and Shawler et al. (1997) Advances in Pharmacology 40: 309-337, Academic Press, Inc., San Diego, Calif.
TRAs, like other antigenic epitopes, are presented at the surface of tumor cells by MHC molecules and have been shown to induce a CTL response in vivo and in vitro. See, for example, van der Bruggen et al. (1991) Science 254: 1643-1647. However, such TRA-expressing tumor cells do not provoke reliable anti-tumor immune responses in vivo that are capable of controlling the growth of malignant cells. Boon et al. (1992) Cancer Surveys 13: 23-37; T. Boon (1993) Int. J. Cancer 54: 177-180; T. Boon (1992) Advances Cancer Res. 58: 177-209. Thus, generation of CTL clones that recognize specific TRAs provides a powerful tool for tumor therapeutics. The identification of TRAs also allows the design of recombinant vaccines for the treatment of various pathological conditions.
The present invention provides isolated CTL clones that are specific for MAGE-1 and MAGE-4, respectively. These CTL clones have been isolated by successive steps of stimulation and testing of lymphocytes with antigen presenting cells which present antigens derived from different expression system, e.g., from recombinant Yersinia or recombinant viruses. The MAGE-1 and MAGE-4 antigenic peptides which are recognized by these CTL clones, as well as the presenting MHC molecules, have also been identified by the present invention.
The present invention relates to isolation of CTL clones. The present invention further relates to newly isolated CTL clones, tumor associated antigens and peptide/HLA complexes.
One embodiment of the present invention provides methods for generating specific CTLs in vitro or ex vivo by successive stimulations and tests of lymphocytes with antigen presenting cells which present antigens derived from different expression systems.
More specifically, the present method for isolating CTL clones includes multiple steps of stimulation and testing with antigen presenting cells. The antigen presenting cells in different steps of stimulation can differ from one another in cell types and in the manner by which the antigen is derived. The use of different antigen presenting cells as stimulator cells reduces CTL activities that are non-specific for the protein of interest, and thus, permits more efficient isolation of specific CTL clones.
In accordance with the present invention, the antigen can derive from a protein expressed from recombinant Yersinia, recombinant Salmonella, or recombinant viruses, respectively. The recombinant virus can be made using strains of vaccinia, canarypox virus, other pox viruses, adenovirus, herpes simplex virus and the like.
The protein against which specific CTL clones are generated can be a tumor associated protein, an antigenic protein of a pathogen, or the like. More preferably, the protein is MAGE-1 or MAGE-4.
In another embodiment, the present invention provides CTL clones generated by using the method of the present invention. Specifically, the present invention provides three newly isolated CTL clones, clone 462/F3.2, which specifically recognizes a MAGE-1 peptide (230-238) presented by HLA-Cw3; clone 456/H7.11, which specifically recognizes a MAGE-1 epitope (258-266) presented by HLA-B53, and clone H4/13, which specifically recognizes a MAGE-4 epitope (230-239) presented by HLA-A2.
In still another embodiment, the present invention provides isolated antigenic peptides, DPARYEFLW (MAGE-1 258-266) (SEQ ID NO:42) and GVYDGREHTV (MAGE-4 230-239) (SEQ ID NO:44), as well as nucleic acid sequences encoding thereof.
In another embodiment, the present invention provides isolated peptide/HLA complexes, peptide SAYGEPRKL (MAGE-1 230-238) (SEQ ID NO:2) complexed with HLA-Cw3, peptide DPARYEFLW (MAGE-1258-266) (SEQ ID NO:42) complexed with HLA-B53, and peptide GVYDGREHTV (MAGE-4 230-239) (SEQ ID NO:44) complexed with HLA-A2. Cells expressing any of the peptide/HLA complexes are also contemplated.
The isolated CTL clones, the antigenic peptides, the peptide/HLA complexes, and cells expressing the peptide/HLA complexes of the present invention can be used in pharmaceutical compositions as well as methods for diagnosing and treating various pathological conditions.